This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Dengue is the most common vector-borne viral disease in humans. Because of the lack of vaccines and drugs, emerging and resurging of dengue have become increasing threats to human health in tropical and subtropical regions. Dengue virus (DENV), a positive-sense, single-stranded RNA virus, is transmitted to humans through the bite of infected Aedes mosquitoes, including Ae. aegypti and Ae. albopictus. Understanding the molecular interactions between dengue virus and the vector mosquito is the prerequisite for developing effective strategies for vector control. However, little is known about how mosquitoes cope with virus infection and what the innate immunity against viruses is. RNA interference (RNAi) is a small RNA-mediated gene-regulatory mechanism that is conserved in most eukaryotic organisms. In plants and Drosophila, RNAi has been identified as a natural antiviral immunity. Viruses are both initiators and targets of RNAi, and replicating viral RNAs are recognized by RNAi machinery and processed into viral small interfering RNAs (siRNAs), which direct specific degradation of homologous viral RNAs. On the other hand, viruses encode proteins to suppress RNAi-based antiviral defense to achieve successful infection. In Aedes mosquitoes, engineering RNAi that targets DENV sequences could lead to resistance to DENV infection or inhibition of virus replication and accumulation. However, it's still not clear in mosquitoes if RNAi mechanism functions as a major natural antiviral immunity, and how viruses interact with RNAi pathway. The objective of this project is to investigate the natural role of RNAi in Aedes mosquito cells in response to DENV infection. Specific Aim 1. Characterization of small RNAs in DENV-infected Aedes mosquito cells In plants and Drosophila, RNAi-based antiviral defense can be induced by virus infection, which is correlated with accumulation of abundant siRNAs derived from the silenced viral genome. Viral siRNAs are considered as a molecular signature of the antiviral RNAi in infected cells. (1) Determine if DENV viral siRNAs accumulate in infected Aedes mosquito cells (2) Perform ultra-deep sequencing of small RNAs from DENV-infected Aedes mosquito cells (3) Identify and characterize small RNAs in DENV-infected Aedes mosquito cells Specific Aim 2. Identification of RNAi suppressors encoded by DENV Besides the detection of viral siRNAs in infected cells, identification of RNAi suppressors encoded by DENV is another direct evidence to support that RNAi serves as natural antiviral immunity in Aedes mosquito cells. (1) Determine if DENV infection can inhibit RNAi in Aedes mosquito cells (2) Identify DENV proteins that can inhibit RNAi in Aedes mosquito cells (3) Determine if DENV proteins can suppress RNAi in mammalian cells (4) Perform mechanistic analysis of RNAi suppression by DENV proteins